The present invention relates in general to apparatus and methods for applying reagents to and removing reagents from slides. In particular, the present invention relates to apparatus and methods for immunohistochemical staining of biological material on glass slides using economically small amounts of reagents.
Histology refers to the study of the microscopic structure of cells, tissues, and organs. The traditional method of analyzing the microscopic structure of an organ is to cut extremely thin sections of tissue on a special device called a microtome. After embedding the tissue in paraffin, a microtome can cut it 3-5 microns thick. Next, the section of tissue is attached to a glass slide. The thin tissue section is then stained with dyes or chemicals which are chosen for their ability to enhance various features in the tissue specimen.
Different dyes may be used to highlight different structures of cells or organs. For example, hematoxylin is a dye which stains cell nuclei purple, and eosin, often used in combination with hematoxylin, is a chemical compound which stains the cell cytoplasm red.
The staining process typically involves dipping the slides into small vats or jars containing the chemicals and/or dyes. Each vat or jar typically contains 0.2-2.0 liters.
More recently, a new type of staining process using specific antibody or DNA probes has been developed. These procedures have the advantage of high specificity and sensitivity, and are commonly used as an adjunct to traditional staining procedures. However, because the probes are far more expensive than dyes or stains, the probes are applied by a special technique which uses only several drops (about 50-200 microliters) of reagent.
In a common procedure for antibody-mediated staining ("immunohistochemistry"), frozen or paraffin-treated thin tissue sections (about 3-5 microns in thickness) are placed on glass slides. Frozen tissue sections are placed in acetone for temporary storage and are washed in buffered saline before use. Paraffin-treated tissue sections are baked at 56.degree. C., deparaffinized in xylene, rehydrated in decreasing grades of ethanol and treated for 30 minutes with dilute hydrogen peroxide. The tissue sections are washed with buffered saline before use.
Next, each slide is blotted dry without disturbing the thin tissue section. Each slide is then placed in a humidified box, tissue side up. An appropriate first reagent is pipetted onto the tissue in a volume of 50-200 microliters. After the reagent has been added to each slide, the humidified chamber is closed, lest the reagent evaporate and leave the tissue dried and, therefore, spoiled.
After the appropriate incubation period (usually 30-60 minutes) each slide is removed from the humidified box and placed in a slide rack. The rack is immersed in buffered saline which is periodically replaced. After washing sufficiently to ensure that the first reagent is removed, each slide is manually removed from the saline, carefully dried and placed into the humidified chamber. A second reagent is then pipetted onto each slide and the slides are again incubated for 30 minutes. This procedure is repeated until all the incubation steps have been completed (usually 3-4 times).
A severe drawback of the present technique is that it is labor intensive. Steps which require individual manual handling of the slides (such as drying and transferring steps) are tedious and time consuming. Handling each slide at each incubation step (transferring a slide from the slide rack to the humidified box, drying the slide, and replacing the slide back in the rack) requires approximately one minute. For a typical run of 50 slides with four incubation steps each, this translates into 3.3 hours of technician time in addition to the time required for incubation and washing (about 45-75 minutes for each step).
An additional problem with the present technique is that a relatively large inequality of incubation time occurs when about 20 or more slides are stained. Because a significant amount of time is required to individually handle each slide, 15-20 minutes may elapse from the time the incubation of the first slide is begun until the incubation of last slide is initiated. Because the second to fourth incubation steps are typically only about 30 minutes in duration, a 50% disparity in incubation times results. This disparity diminishes the reliability of positive and negative control slides and makes comparisons between slides less valid.
In one approach to the control of the flow of fluid reagents, Thiers, U.S. Pat. No. 3,607,082 discloses a fluid-processing apparatus and a method for its use. In Thiers, metering and valving means charge a plurality of separate liquids into separate pipet chambers and discharge the liquids from the pipet chambers so that each succeeding liquid passes through the pipet chamber employed to receive the preceding liquid. A fluid receiving and transporting conduit has tractable wall portions adapted to receive the metered liquids from the pipets, means for forming movable seals in the conduit to isolate a metered quantity of liquid in a hydraulically isolated chamber, means for mixing the fluids in the chamber by compressing and releasing portions of the wall thereof, processing means for performing various unit processes on the fluid mixture in a given chamber and discharge means for removing fluid mixture from the conduit. A branched-arm valve, fitted with means for selectively closing arms thereof is employed as a valving means in charging and dispensing fluids into and from the pipet chambers. The system is described for use in carrying out various processes of chemistry, microbiology or the like. However, Thiers does not disclose a solution to the problems of applying a small amount of expensive reagent to and removing the reagent from a slide.
Reunanen, U.S. Pat. No. 3,754,863, discloses a method and apparatus for dosing reagents and for incubating and sampling a reaction mixture. In the method, reagents are drawn into reaction syringes in which an incubation is carried out and from which samples are discharged in one or more portions. The apparatus consists of a syringe unit, reaction syringes mounted on a single frame, syringes containing stopping liquid and an internal standard. Vessels for the reagents, instruments for feeding reagents and for the automatic adjustment of pistons and for transferring absorption plates, and a thermostat are mounted on a fixed outer frame. The invention is indicated to be particularly useful for macromolecular investigation of radioactive parent substances where the sample is absorbed into a porous medium and where it is washed automatically. Nevertheless, Reunanen does not disclose methods or apparatus particularly suited to manipulation of small amounts of reagent materials for application to tissue sections of the sort which are employed in immunohistochemistry.
In another approach, Wallach, U.S. Pat. No., 3,764,215, discloses a medical specimen, such as a Pap smear, blood or bacteria sample, placed on a thin transparent flexible slide. Several slides are affixed to one another or to an elongated flexible carrier and then wound on a reel. A plurality of such reels are loaded on top of one another into a vertical processing tank which is filled sequentially with chemicals and stains as required. Subsequently, the reel is removed from the tank and the slides are microscopically screened to detect diseases such as cancer and the like. However, the expense of immunohistochemical reagents and the degree of care with which they must be applied render impractical the dipping of reels of slides into tanks of reagent.
In Becker et al., U.S. Pat. No. 3,837,795, a method for staining biological material on flat slides is disclosed. A group of slides is successively dipped into vessels containing different liquids conventionally used in staining. The time a group of slides is in any given vessel is controlled in accordance with the staining technique desired. One of the vessels has a de-colorizing agent in it. In this last vessel, a laminar flow of de-colorizing liquid is directed over the flat faces of the slides to create a chimney effect which minimizes cross-contamination. The de-colorizing liquid is withdrawn continuously from its vessel, filtered and re-introduced into the vessel. However, because Becker et al. teaches the dipping of slides into vessels, the approach of Becker et al. suffers from the disadvantages common to techniques where large quantities of reagent are employed.
Hastka, European Patent Publication No. 201,780, described an apparatus for histological examination which has a plurality of chambers, each chamber having wall delineating apertures and having a system of a magnet and a magnetically attracted material to hold each chamber to a slide. Each chamber is said to have an outlet conduit with a valve connected to it, and these outlet conduits are reported to be connected by a vacuum source by way of a manifold. Rasmussen, U.S. Pat. No. 4,358,478, describes a closed chamber with an inlet and an outlet port, the inlet port being connected to a valve through which fluid from a reservoir is reported to be fed. However, Hastka does not suggest an inlet port for applying materials to a chamber reversibly sealed to the surface of a slide, and although Rasmussen discloses an inlet port, it is a port to a chamber which is not reversibly sealed to the surface of a slide. Therefore, neither Rasmussen nor Hastka suggests an apparatus or method to provide histological slide-staining chambers with a controllable flow of reagent or rinsing solution for the treatment of biological samples on slides in a fashion useful for reproducible staining of large numbers of slides.
Cytochemistry apparatus is disclosed in Brigati, a presentation made at the Massachusetts Society for Histotechnology 13th Annual Seminar, Apr. 23-25, 1987, in Eastham, Mass. A similar but less detailed presentation was made at the Spring Meeting of the American Society of Clinical Pathologists, Mar. 28-Apr. 2, 1987, San Francisco, Calif. Brigati discloses an apparatus for immunocytochemistry which employs a capillary gap to facilitate application and removal of small amounts of reagents. Specifically, a glass slide with a protruding spacer shim is closely opposed to a slide to which a thin tissue sample is attached, thus forming a capillary gap between the slides. The gap between the opposed slides is alternately passed over reagent-filled recesses in a hydrophobic surface or over a blotter to respectively introduce or remove a reagent. Nevertheless, the reliance upon capillary action to properly distribute reagent to the surface of a tissue sample may affect the uniformity of distribution (e.g. due to the formation of bubbles in the capillary space) and limits the thickness of tissue samples which may be used. Moreover, special slides having a painted spacing shim are required. Another disadvantage of this technique is that the tissue section must be applied to the slide near the bottom end. If the tissue is too far up, the reagent may not climb sufficiently by capillary action to immerse the tissue section.
Therefore, there is still a need for an efficient and rapid yet simple apparatus for immunohisto-chemical staining.